1. Field of the Invention
A considerable portion of world-wide research efforts in the treatment of cancer is currently devoted to killing cancer cells by means of various cell killing agents. Despite the fact that numerous drugs, radioactive compounds, and the like have been shown to be capable of killing cancer cells, these agents fail to treat cancer successfully because of their inability to circumvent three universally present obstacles: (1) the agents do not kill all the cancer cells because they do not exhibit cytotoxic specificity for all the cancer cells, (2) the agents also kill normal cells because they do not exhibit cytotoxic specificity exclusively for cancer cells, and (3) the agents are not potent enough to kill resistant cancer cells or to overcome the ability of cancer cells to adapt and become resistant to the cell killing agents. An appreciation of these three obstacles is necessary to understand why current treatments fail and to understand the rational and methodology of the proposed invention.
Fifty years of intense research has shown that there is a wide heterogeneity in every characteristic that has been measured in cancer cells. These characteristics include cell size, buoyancy, anaerobic metabolism, enzyme composition, growth rate, gene errors, differential gene expression, chromosome number, and chromosome errors. The heterogeneity is also expressed by the presence of some cancer cells that are super-sensitive and others that are super-resistant to being killed or treated by any therapeutic agent. Within the same tumor population a fraction of cells will be sensitive to a given therapeutic agent and will be killed when that agent is administered, a fraction of cells will be resistant to the agent and will not be killed, and a fraction will adapt and become increasingly more resistant to subsequent therapeutic regimens. The resistant cells will continue to divide and spread to distant locations in the body to form metastatic tumors.
The wide heterogeneity in sensitivity to any particular therapeutic agent leads to the high probability that the systemic application of any therapeutic agent will cause partial remission of the tumor by killing the super-sensitive cancer cells, but will not be able to achieve a complete cure because it cannot kill the super-resistant cancer cells. Previous attempts at cancer therapy have generally ignored the negative therapeutic consequences of these divergent cells. There has been an intuitive and optimistic belief than an approach achieving partial remission in its early phase will give a complete cure after it has been fine-tuned. This optimism contradicts the biological principle, supported by a large amount of data, that every large population of cells or organisms is heterogeneous, and that cancer cells, which have a genetic instability, exhibit a particularly high degree of heterogeneity. Therefore, it is not surprising that the past history of cancer therapy approaches has been a monotonous sequence of short periods of hope, because killing some cancer cells leads to a remission, followed by prolonged periods of disappointment, because some cancer cells survive, seed, and continue to grow in the living host and subsequent treatments are less effective at killing the cancer cells of these metastatic tumors. It is likely that the latest field of oncogenes and other gene manipulations, as applied to cancer therapy, will also follow the same pattern. This prediction is based on the fact that there is a heterogeneity of gene errors and gene expression in the cancer cell population, and with time, more and more cells, with more and varied genetic and chromosome errors accumulate in the cancer cell population. No simple genetic correction, even if it could be applied successfully to all the cancer cells growing in the body, is likely to repair every cell.
2. Prior Art
The first serious deficiency of current cancer therapeutic approaches is that they do not take into account, and are unable to deal with, the heterogeneity of the cancer cell population. The inability of current approaches to circumvent this heterogeneity is illustrated by the failure of immuno-therapeutic approaches that rely on antigenic receptors on the surface of cancer cells to deliver therapeutic agents.
All current attempts at cancer therapy (apart from the treatment of thyroid cancer with radio-iodide) depend on killing each and every individual cancer cell by their direct individual interaction with the candidate therapeutic agents or applied environmental condition. In order to describe the need for this direct interaction, these strategies can be loosely called "sniper killing," i.e. each cell to be killed must be targeted directly. Sniper killing agents include cytotoxic drugs, binary reagents made by attaching cancer targeting agents to cytotoxic drugs, augmented immune response, hormonal therapy, genetically engineered products (like interferon), manipulations of oncogenes, or products coded by these genes.
In order for these sniper killing strategies to be successful in treating cancer, it would be necessary for the cancer cells to have an exploitable characteristic which is present on all cancer cells, for that characteristic to be absent from all (or at least most) normal cells, and for that characteristic to not adaptively change and become non-exploitable.
It is known that cancer cells exhibit on their surface numerous receptors, including antigenic receptors, to which selected molecules such as specific antibodies, hormones, and peptides can bind. Antibodies, hormones, and peptides can be used as targeting agents for the cancer cells that express those particular antigenic receptors. Ideally, all cancer cells would express the receptor, and the number of non-cancerous cells which express the receptor would be very small. In the ideal model, binary reagents (an example of a sniper strategy) which are composed of targeting agents and cytotoxic agents would be preferentially directed to the cancer cells. However, in practice, binary reagents do not result in the delivery of the cytotoxic agent to all cancer cells in the tumor population because some cancer cells do not exhibit the particular antigenic receptor. The binary reagent will not attach to these antigenic receptor deficient cancer cells, and therefore these cells will be unaffected by the treatment and will be left to proliferate in the host. High-dose sniper killing, even when employed at dose levels which kill many normal cells, fail to kill all cancer cells because some cancer cells are antigenic receptor deficient, some cancer cells are super-resistant even before the treatment begins, and some cancer cells adapt to the therapeutic agent, survive, and become resistant to future treatments. All these sniper strategies have failed, and are doomed to fail in the future, because they cannot deal with the fact that some normal cells also express the characteristic which is the target for the sniper killing, and because they cannot deal with the universally present heterogeneity and adaptive ability of cancer cells.
The recent development of highly pure and highly immuno-specific monoclonal antibodies, hormones, and peptides which can act as specific targeting agents for particular antigenic receptors has greatly increased the ability to direct cell killing agents specifically to cancer cells and thereby minimize any adverse effects on non-cancerous cells. Paradoxically, this current direction of isolating and producing such highly specific targeting agents (for the purpose of minimizing the possibility that such antibodies, and the cytotoxic agents carried thereby, might attach to non-cancerous cells) is, in one sense, counter-productive, since the number of cancer cells within the tumor population which will exhibit an affinity for such highly specific targeting agents will be reduced.
Notwithstanding the above-mentioned advances in the development of highly specific targeting agents to deliver the cell killing agents specifically to targeted cells, and the demonstrated cell killing ability of the particular delivered agents, therapeutic success through the use of binary reagents composed of targeting agents and toxic agents has not been achieved, and should not have been expected. Unfortunately, in practice these therapies have been far less successful than they were hoped to be.
The second serious deficiency of binary reagents to carry cytotoxic agents to target cancer cells is that the so called "cancer targeting agents" of which the binary reagents are made, also target a significant number of normal cells. These targeted normal cells are also killed by the administration of binary reagents, cause unacceptable destruction of normal tissues, serious illness of the patient, and limit the aggressiveness of the attack which can be launched against the cancer.
The third serious deficiency of binary reagents to carry cytotoxic agents to target cancer cells, particularly cytotoxic radioactive isotopes, is that they cause significant systemic toxicity because the targeting agent carrying the cytotoxic agent is a large molecule which causes them to have a long residence time in the blood circulation, and causes them to be taken up non-specifically by normal cells.
The fourth serious deficiency of binary reagents to carry cytotoxic agents to cancer cells is that even those cancer cells which the targeting agents attaches to, outright killing of the cancer cell is often not accomplished. In large part this is due to the inherent limitations of the treatment method, i.e., the absolute quantity of cytotoxic agent which can be coupled to the targeting agent is smaller than that required to actually kill the cancer cell (the small quantity of cytotoxic agent which can be attached is limited to avoid destroying the targeting ability of the targeting agent and to avoid adversely altering the distribution of the binary reagent in the host). While the amount of cytotoxic agent which can be brought to bear on cancer cells through the use of binary reagents may be sufficient to damage some of the cells, the damage often is only temporary or, indeed, simply results in the emergence of mutant cells which are still cancerous and have become resistant to the effects of the cytotoxic agent.
The fifth serious deficiency of the binary reagents to carry cytotoxic agents is that it is impossible to make a valid choice of the most appropriate targeting agent to make the binary reagent for each cancer in each patient. Furthermore, it is not possible to predict the outcome of the therapy prior to administering the binary reagent at the necessary cytotoxic dose level.
Despite the three obstacles and the deficiencies described above, the treatment of thyroid cancer with radio-iodide is successful in a high proportion of cases. This high rate of success is not due to a fundamental difference between cancer cells of the thyroid and cancer cells which have arisen from other tissues. The successful treatment of thyroid cancer is due to the fact that normal and malignant thyroid cells have a unique biological function which allows them to store iodine. Thus, when patients with thyroid cancer are treated with radio-iodide, a fraction of the cancer cells take up sufficient quantities of isotope and store the isotope long enough to generate micro-regions of intense radiation in which all the cells in each micro-region are killed. These intense radiation fields, called Hot-Spots, are generated exclusively in the normal and malignant thyroid tissue. The radiation field in the Hot-Spots extends beyond the cells taking up the isotope and kills hundreds of neighboring cells thereby creating overlapping micro-regions of supra-lethal radiation (overlapping Hot-Spots) exclusively in the thyroid tissue. Inside these Hot-Spots, the radiation is so intense that all the cancer cells in the tumor are killed, including the cells that do not take up the radio-isotope.
Two types of strategies have been employed to amplify and localize the effect of cytotoxic agents on targeted cells in order to circumvent the five deficiencies described and in order to simulate the operating conditions that make the treatment of thyroid cancer so successful. The first strategy attempts to accumulate the cytotoxic agents inside targeted cells and the second strategy attempts to form and accumulate cytotoxic agents outside targeted cells in the extra-cellular fluid of the tumor.
The first strategy to amplify and localize the effect of cytotoxic agents on targeted cells is to accumulate cytoxic agents inside cells. In many normal and disease states, it is desirable to target therapeutic and/or tracer chemicals to specific cell types. Two problems exist in such targeting. The first problem is how to cause the targeting to be specific for certain cell types. The second problem is how to accumulate and retain the therapeutic and/or tracer chemical in the region of the targeted cells for as long as possible in order to maximize the effect on the targeted cells, and at the same time minimize the effect on non-targeted cells by preventing the therapeutic and/or tracer chemical from leaving the region of the targeted cells, diffusing away, and reaching the regions of non-targeted cells.
Progress has been made on the first problem by accumulating the therapeutic and/or tracer agent inside targeted cells. This has been achieved by constructing a binary reagent by covalently attaching the therapeutic or tracer chemical to proteins, such as antibodies, hormones, or peptides which act as targeting agents (Ghose T. and Blair A. H. 1987, CRC Critical Reviews of Therapeutic Drug Carrier Systems, 3, 262-359; Blakely et al. 1988, Progress in Allergy, 45, 50-9). The protein targeting agent moiety of the binary reagent binds to endocytosing antigenic receptors on certain cell types, called target cells, and delivers the therapeutic or tracer chemical agent to the desired target cells. The binding of the targeting agent to the antigenic receptor on the target cells induces the target cells to undergo receptor mediated endocytosis which causes the cells to "swallow" the receptor and bound binary reagent, and to transport the receptor and binary reagent to lysosome vacuoles. The lysosome vacuoles have an acidic environment and contain a high concentration of numerous proteolytic, glycanolytic, nuclease, and lipolytic enzymes. Once inside the lysosomes, the receptors are released from the binary reagents and recycle back to the cell surface to bind more binary reagents and to thus continue repeating the receptor mediated endocytosis process. In this manner, each receptor can recycle 5 to 10 times per hour. Inside the lysosomes, the targeting agent moiety of the binary reagent is digested, and the therapeutic or tracer chemical is released as a free, soluble molecule. In this free state the chemical exerts its tracer or pharmacological therapeutic action.
Cytotoxic drugs, toxins, dyes, antidotes to toxic drugs, and molecules carrying radio-isotopes have been delivered to cells by this means (Ali et al. 1990, Cancer R. Suppl. 50, 783-788; Wu et al. 1985, Hepatology 1985, 5, 709-713; Wu et al. 1983, Proc. Nalt Acad. Sci., 80, 3078-3080; Firestone Raymond 1994, Bioconjugate, 5, 105-113; C. Rushfeldt and Brad Smedsrod 1993, Cancer Research 1993, 53, 658-662; Pittman et al. 1983, Biochem. J. 212, 791-800; Jansen et al. 1992, Hepatology 18, 146-152; Daniel A. Vallera 1994, Blood, 83, 309-317; A. Mukhopadhyay and S. K. Basu 1990, Biotechnology and Applied Biochem. 12, 529-536).
Some progress has also been made regarding the second problem of the attached therapeutic and/or tracer chemical leaving the targeted cells . The second problem has been partly solved by trapping the released chemical in the lysosomes of the targeted cell. For example, one approach to the problem of intra-cellular trapping that has been described uses a common disaccharide, sucrose, as a marker of fluid endocytosis. Since mammalian cells lack the necessary glycosidase, the sucrose is not digested, and since sucrose is unable to rapidly cross the cell membrane, the sucrose is partially trapped in the cell. Thus, the amount of sucrose which is trapped can be used as an approximate measure of sucrose uptake.
Taking advantage of these properties of sucrose, a technique was developed for determining the sites of degradation of plasma proteins, by using the proteins as targeting agents which are covalently attached to radio-sucrose to make a binary reagent The binary reagent is introduced into targeted cells by receptor mediated endocytosis to measure the rate of degradation of the targeting agent protein. After the administration and receptor mediated endocytosis of the binary reagent, the protein targeting agent moiety of the binary reagent is enzymatically digested, causing the release of the soluble radio-sucrose molecules as a free molecules. Since sucrose is not degraded and remains partially trapped within the cell, the amount which has accumulated in the cell can be used as an approximate measure of the amount degradation of the protein targeting agent by the targeted cells (Pittman and Steinberg 1978, Biochem. Biophys. Res. Commun. 81, 1251-259; Pittman et al. 1979, J. Bio. Chem.; 254, 6876-6879; Pittman et al. 1979, Proc. Natl. Acad. Sci. USA 76, 5345-5349).
More recently, it has been shown that soluble cellobiose can be used in a similar manner to sucrose. Cellobiose can be linked by a non-metabolizable bond to the therapeutic or tracer chemical, so that the soluble cellobiose and the attached therapeutic or tracer chemical, once free from its attachment to the targeting agent, accumulates in the targeted cells (Pittman et al. 1983, Biochem. J. 212, 791-800; Pittman, 1984, U.S. Pat. No. 4,466,951). The cellobiose method has certain advantages over the use of sucrose.
Nevertheless, both the soluble sucrose and cellobiose have the disadvantage in that the accumulated carbohydrate, with or without an attached therapeutic chemical, slowly leaves the cell. Therefore, cells cannot continue to accumulate increasing amounts of carbohydrate. There is the added disadvantage that the accumulated carbohydrate can diffuse away from the targeted cell and reach cells which were not targeted.
The second strategy to amplify and localize the effect of cytotoxic agents on targeted cells is to form the cytotoxic agents outside targeted cells in the extra-cellular fluid. The formation of cytotoxic agents outside targeted cells in the extra-cellular fluid of the targeted regions has been achieved by the enzymatic conversion of a pro-drug into an active drug by a method called Antibody Dependent Enzyme Pro-Drug Therapy (ADEPT). The enzyme which makes the conversion is one moiety of a bispecific reagent, the other moiety being an antibody with a binding affinity to the non-endocytosing receptors on surface of targeted cancer cells. Since the enzyme moiety is bound to the surface of the targeted cells, the conversion from pro-drug to the active drug takes place in the extracellular fluid.
The active drug diffuses into the immediate micro-region to have its pharmacological cytotoxic effect on the non-target cancer cells in the micro-region. For example, alkaline phosphatase converts the pro-drugs mitomycin phosphate into an active mitomycin C derivative and etoposide phosphate into an active etoposide (Senter et al, 1989, Cancer Research, 49, 5789-5792), beta-lactamase converts a cephalosporin derivative of 4-de-succetylvinblastine-3-carboxhydrazide into an active cytotoxic drug (Meyer et al, 1993, Cancer research, 53, 3956-3963), and activates cephalo- doxorubcin (Rodrigues et al, 1995, Cancer Research, 55, 63-70), DT diaphorase followed by a non-enzymatic reaction with a thioester activates the mono-functional alkylating agent CB 1954 into an active agent which can cause ctotoxicity by cross-linking DNA (Knox et al, 1993, Cancer and Metastasis Reviews, 12, 195-212); carboxypeptidase G2 can convert a nitrogen mustard prodrug into an active drug (Springer and Niculescu-Duvaz, 1995, Anticancer Drug Des. 10, 361-372); nitroreductase can activate CB1954 (Knox et al, 1995, Biochem. Pharmacol., 49, 1641-1647); and dinitrobenzamide (Anlezark et al, 1995, Biochem. Pharmacol., 50, 609-618); to form cytotoxic derivatives, and alpha-galactosidase can activate prodrugs of anthaacycline (Azoulay et al, 1995, Anticancer Drug Des., 10, 441-450).
The three step ADEPT approach fails to successfully treat cancer for the following reasons: (a) the bispecific reagent is bound to the non-endocytosing target cancer cells and also to some normal cells because the targeting agent moiety does not exhibit exclusive cytotoxic specificity for cancer cells which reduces the tumor specificity of the non-mammalian enzyme location and pro drug conversion, (b) the antigenic receptors of the target cells are in a constant state of flux which prevents the bispecific reagent from remaining bound for a sufficient period of time to allow all bispecific reagent not bound specifically to the target cell receptors to be eliminated from the body prior to administering the pro-drug; (c) the soluble active drug which is made by the enzyme diffuses away from its site of production to have a cytotoxic action on healthy normal cells; (d) the cells on which the bispecific reagent is bound, and where the active drug is formed, are the first cells to be killed because they receive the highest concentration of the active drug. When these cells are killed, the enzyme will no longer be in a position to convert the pro-drug into an active drug and,therefore, the production of active drug is self limiting; and (e) the shape and volume of the micro-region in which there is a sufficiently high concentration of the active drug to kill cells is variable and ill-defined because the diffusion parameters of the soluble active drug are dependent on the particular status of the blood capillaries and extra-cellular structures in the cancer, the parameters of the diffusion varying from one location of the tumor to another.
The two strategies described above fail to generate Hot-Spots because the number of cytotoxic chemical or radio-isotope agents which are delivered is small, the number being directly proportional to the relatively small number of antigenic receptors on the surface of the target cells. In addition, the agents or isotopes do not remain in the correct location for long enough to achieve an aggressive attack on the cancer, and furthermore, they cause systemic toxicity because the agents circulate in the blood for a long period of time. Finally these strategies also fail to locate the attack specifically to the tumor, because the location where the agent or isotopes are delivered or where the active drug is made is dependent on only a single cancer associated characteristic on the cancer cell surface, and every single characteristic found on cancer cells is also found on some normal cells.
The present invention mimics for non-thyroid cancers, the Hot-Spot killing which makes the treatment of thyroid cancer successful; however, since no other malignant tissue has the same natural iodide involving process as the thyroid, the mimicking requires the construction of a special, multi-step, sequential process to achieve "Hot-Spots" in non-thyroid cancers. The basic process of the present invention consists of sequential steps which act independently and together with naturally occurring characteristics of the cancer and normal cell populations to generate overlapping Hot-Spots virtally exclusively in the tumor without causing significant systemic toxicity. Cancer cells within these Hot-Spots are eradicated, the eradicated cells include cancer cells that are not targeted, cancer cells that are resistant and even super-resistant, and cancer cells that would otherwise adapt and become resistant to therapy.